Peptides play a pivotal role in vivo because of their ubiquitous involvement in a variety of interactions with other biological molecules and proteins such as cell receptors, antibodies or enzymes. Although they are naturally flexible and can adopt a large number of different conformations, their biological activity is usually the result of one specific three-dimensional conformation. In laboratory experiments the conformation of peptides is also studied to reproduce specific determinants (epitopes) of high molecular weight proteins. The length of the peptides used for these studies is in general limited (up to fifteen amino acids) to overcome the problems that arise with increasing length of the peptide.
However, these peptides show a higher flexibility compared to the native protein. For this reason, their biological activity is usually lower than in the native conformation unless they are in some way rigidified. One way to achieve this is to synthesize different head-to-tail cyclic peptides with restricted conformational flexibility and to determine which of these analogs retains biological activity (Kessler H., 1982, Angew. Chem. Int. Ed. Engl., 21, 512-523; Kessler H et al., 1989, in Computer-aided drug design, methods and applications, Ed T. J. Perun and C. L. Probst, pp. 461-484, Marcel Dekker, New-York; Hruby V. J. et al., 1990, Biochem. J., 268, 249-262; Toniolo C., 1990, Int. J. Pept. Protein Res., 35, 287-300; Gilon C et al., 1991, Biopolymers, 31, 745-750).
Cyclic peptides can be formed in solution, in diluted conditions, by activation of a side-chain protected linear peptide using carbodiimide or diphenylphosphorylazide, followed by subsequent deprotection of the side-chains (Kessler H et al., 1989, in Computer-aided drug design, methods and applications, Ed. T. J. Perun and C. L. Probst, pp. 461-484, Marcel Dekker, New-York; Toniolo C., 1990, Int. J. Pept. Protein Res., 35, 287-300; Gurrath M. et al., 1992, Eur. J. Biochem., 210, 911-921; Izumiya N. et al., 1981, Biopolymers, 20, 1785-1791; Brady S. F. et al., 1983, in Peptides, Structure and Function, Proceedings of the Eighth American Peptide Symposium, Ed. V. J. Hruby and D. H. Rick, pp. 127-130, Pierce Chemical Company, Rockford, Illinois; He J. X. et al., 1994, Lett. Peptide Sci., 1, 25-30). Side-chain to side-chain cyclization has been achieved through lactam (Hruby V. J. et al., 1990, Biochem. J., 268, 249-262; Hoffmann E. et al., 1991, Liebigs Ann. Chem., 585-590) or disulfide bridge formation, between two cysteines incorporated in the sequence (Hruby V. J. et al., 1990, Biochem. J., 268, 249-262; Cavelier F. et al., 1989, Bull. Soc. Chim. France, 788-798). Cyclization of unprotected peptides has also been obtained by oxime formation in aqueous solution (Pallin T. G. and Tam J. P., 1995, J. Chem. Soc., Chem. Commun., 2021-2022). Although these methods have been widely used, they suffer from the fact that they are time-consuming and that side reactions like cyclodimerization, oligomerization or racemization of the C-terminal residue are difficult to avoid.
To overcome this problem, a fully automated synthesis procedure has been proposed, with the cyclization step occurring directly on the column (Kates S. A. et al., 1993, Anal. Biochem., 212, 303-310). This method allows the synthesis of cyclic peptides containing an acid (Asp, Glu) or an amide residue (Asn, Gln) (Kates S. A. et al., 1993, Anal. Biochem., 212, 303-310). In this method, an .alpha.-allyl protected acid residue like Fmoc-Asp-OAl or Fmoc-Glu-OAl is incorporated as the first residue during the synthesis by fixation of its free carboxylic side-chain on the synthesis support. The peptidic chain is then elongated following the standard Fmoc synthesis procedure before specific cleavage of the .alpha.-allyl protecting group, at the carboxyterminal end of the peptide, with palladium (0). Then the nucleophilic attack of the amino-terminal group of the peptide on the deprotected carboxylic end, allows the head-to-tail cyclization on the column (Kates S. A. et al, 1993, Anal. Biochem., 212, 303-310).
Other strategies incorporating (bromoacetyl) diaminopropionic acid in the sequence (Ivanov et al., 1995, Bioconjugate Chem., 6, 269-277) or bromoacetyl-beta-alanyl-lysine derivatives (U.S. Pat. No. 5,444,150) are also described in the scientific literature. Cyclization was for example obtained by covalent linkage between diaminopropionic acid and a cysteine residue of the sequence. This cyclization method was not automated and was achieved by a side-chain to side-chain link.